Crystal structure of F2F3:NS1A (85-215) complex. (A) Gel filtration data demonstrating complex formation between NS1A (85-215) and F2F3. Traces show chromatographic profiles on a Superdex G75 column for NS1A (85-215) alone (red) and the complex of NS1A (85-215) with [S94]-F2F3 (blue). Inset shows calibrated gel filtration data for (A) [S94]-F2F3 (∼10 kDa), (B) NS1A (85-215) alone (∼27 kDa), and (C) [S94]-F2F3:NS1A complex (∼47 kDa). Similar results were obtained by static light scattering analysis of effluent fractions from size exclusion chromatography, as described in SI Materials and Methods: (A) 15 ± 3 kDa, (B) 25 ± 5 kDa, and (C) 48 ± 5 kDa. The molecular mass expected for the tetrameric complex observed in the crystal structure is 49,250 Da, which is in good agreement with these light scattering and gel filtration data. The elution times for isolated NS1A (85-215) (single chain calculated molecular mass 15,943 Da) and [S94]-F2F3 (single chain calculated molecular mass 8,682 Da) domains differ when loaded at different protein concentrations, suggesting that these molecules form weak homodimers under these solution conditions. Calibration standards (A–D) are described in SI Materials and Methods. (B) Two NS1A effector domains (green and red) and two F2F3 domains (blue and yellow) of CPSF30 form the tetramer. Some NS1A′ amino acid residues that function in complex formation are highlighted in cyan. (C) F3-binding pocket on NS1A (85-215). A hydrophobic pocket on the NS1A surface binds to the F3 Zn finger of F2F3. Both chains of NS1A in the head-to-head dimer interact with each F2F3 molecule. (D) Expanded view of the F3-binding pocket. The NS1A amino acid residues labeled in red interact with the aromatic side chains of residues Y97, F98, and F102 of the F3 Zn finger of F2F3.

Effects of amino acid substitutions in the NS1A protein on its interaction with CPSF30 and on its function in influenza A virus-infected cells. (A) GST-pulldown assay. GST-F2F3 or GST were mixed with equal amounts of the WT or indicated mutant ³⁵S-labeled full-length NS1A protein of Ud, which were prepared as described in SI Materials and Methods. The labeled proteins eluted with glutathione from GST-F2F3 or GST were resolved by SDS-polyacrylamide gels, which were analyzed by exposure to X-ray film. (B) Plaque sizes of the WT and G184R mutant Ud viruses in Madin-Darby canine kidney (MDCK) cells. (C) The G184R mutation in the Ud NS1A protein does not affect the amount of the NS1A protein synthesized in MDCK cells infected with 5 pfu/cell. Immunoblots of cell extracts collected at 6 h after infection were carried out with either anti-NS1A or antitubulin antibody. (D) Quantitative RT-PCR measuring amounts of IFN-β pre-mRNA (Left) and IFN-β mRNA (Right) in WT and G184R Ud-infected cells. Pre-mRNA results were normalized to WT, and mRNA results were normalized to G184R data. The results show the average and standard deviation for the relative levels of G184R pre-mRNA and WT mRNA from three different virus infections.

Structural role of F103 and M106 in formation of the tetrameric complex. (A) Molecular graphics showing how two F2F3 molecules (represented as the electrostatic potential surface) wrap around two NS1A (85-215) molecules. The head-to-head interaction of NS1A molecules forms a docking surface for F2F3 binding. The side chain of residue M106 is critically positioned at the tetrameric epicenter and interacts with the other three molecules. (B) Expanded regions showing the structural environments of amino acid residues F103 and M106 of NS1A. The aromatic side chain of NS1A F103 interacts primarily with the hydrophobic amino acid residues L72′, Y88′ and P111′ that are present on the surface of F2F3′. (C) GST-pulldown assay. GST-F2F3 or GST was mixed with the WT or 103/106 mutant [F103L, M106I]-NS1A protein, and analyzed as described in the legend of Fig. 2A.

Molecular graphic showing the locations of NS1A residues F103 and M106 with respect to the F3-binding pocket. The tetrameric interface extends beyond the hydrophobic pocket of the NS1A effector domain (orange) which binds one F2F3 molecule (blue). Residues M106 and F103 (green surfaces) of the same NS1A effector domain interact with both the F2F3 (blue) and F2F3′ (yellow) molecules. The M106 sidechain of this NS1A effector domain also interacts with the M106′ side chain of the second NS1A′ molecule (red) at the tetrameric epicenter.

PR8 virus infection, unlike infection by Ud virus, does not activate IRF-3. HEL299 cells were either mock-infected (M, lane 1), infected with 5 pfu/cell of Ud virus (Ud, lane 2), infected with 5 pfu/cell of PR8 virus (PR8, lane 3), or infected with 5 pfu/cell of a recombinant Ud virus in which the Ud NS gene was replaced by the PR8 NS gene (Ud/NS-PR8, lane 4). At 7 h after infection, cell extracts were prepared, subjected to electrophoresis on a 7.5% native gel, and IRF-3 monomers and dimers were detected by Western immunoblotting using rabbit anti-IRF-3 antibody (36). An immunoblot with anti-NS1A antibody confirmed that equivalent amounts of the NS1A protein were synthesized in Ud, PR8, and Ud/NS-PR8 virus-infected cells (lanes 2–4). Further details are provided in the SI Materials and Methods.